Ivis 200 system

NOD-scid IL2Rgamma^null (NSG) mice (Jackson Laboratories, 005557) were housed at the Boston Children's Hospital animal care facility following institutional guidelines. 8 to 12-week-old male and female mice were intravenously injected with 1x10⁶ OCI-Ly1 DLBCL tumor cells expressing green firefly luciferase. The inoculated animals were subjected to bioluminescence imaging (BLI) using the IVIS 200 system (PerkinElmer) twice per week following intraperitoneal injections of Vivoglo luciferin (Promega, P1043) at 150mg/kg body weight. After two weeks, animals with substantial tumor cell engraftment (Mean total flux > 5x10⁵ photons/sec) were randomly assigned into four groups and intravenously injected with PBS (untreated) or 2x10⁶CAR T cells generated from control iPSC-SF-T, iPSC-EZ-T, or PBMC-T cells. Tumor burden was measured by BLI weekly, and images were processed and analyzed using Aura imaging software (Spectral Instruments Imaging). To monitor CAR T cell persistence, peripheral blood cells were collected via retro-orbital bleeding after 3 weeks of T cell injections, and absolute numbers of CAR T cells were determined by flow cytometry analysis. All animal experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of Boston Children's Hospital.

Bioluminescence imaging was utilized to assess FLuc activity. D-luciferin (1 mM, Caliper, Hopkinton, MA) was added to wells and plates were incubated at 37°C for 30 min, followed by imaging with an IVIS 200 system (Perkin Elmer, Waltham, MA). For assessing GLuc activity, a GLuc activity kit (New England Biolabs) was used. Media (10 μl/well) was sampled and placed in a black 384-well plate. Substrate solution (20 μl/well) was added and luminescence was measured with a 1 s integration time using a Synergy 4 plate reader (BioTek, Winooski, VT). After each time point, media in wells was partially replaced with the addition of appropriate stimulants and treatments.

1×10⁶ LS174T cells expressing firefly luciferase were delivered intraperitoneally (i.p.). 1×10⁶ NK-92MI cells were labeled using Cy5.5 NHS ester (Lumiprobe Corporation, Hannover, Germany) for 30 min before injection, according to the manufacturer’s instructions, and delivered either by intravenous (i.v.) or i.p. injections seven days post-tumor cell injection. Bioluminescence and fluorescence were determined using an in vivo imaging system (IVIS200 system, Perkin Elmer, Waltham, MA, USA). At study termination, mice were sacrificed and samples of tumors, serums, peritoneal washes, spleens, and livers were collected for further analysis.